1. Field of the Invention
This invention relates to a method for mass calibration, and more particularly a method for mass calibration in a mass spectrometer having an ion trap type mass analysis region.
2. Description of Related Arts
At present, it has been required to establish a technology for performing an analysis of mixture in the field of the analysis technology. For example, in the case that some harmful substances in environment are analyzed, various kinds of substances are contained in the collected samples (for example, water of lake or pond). In addition, a similar state occurs also in the field of analysis of biological compound. It is known that some various kinds of substances are contained in the samples based on the biological compounds such as blood or urine. As described above, in the case that the environmental compounds or biological compounds are analyzed, it is required to provide a technology in which mixtures are processed and analyzed.
Since it is generally difficult to perform a direct analysis of the mixture, each of the components is detected and identified after the desired components in the mixture are separated. In such a circumstance as described above, a liquid chromatograph/mass spectrometer (hereinafter abbreviated as an LC/MS) in which a liquid chromatograph showing a superior separation of mixture and a mass spectrometer showing a superior identification for substances are coupled to each other is quite effective for analyzing some compounds containing several kinds of compounds such as the aforesaid environmental compounds or biological compounds to be analyzed.
Referring now to FIG. 1, the prior art LC/MS using the mass spectrometer having an ion trap type mass analysis region will be described.
A liquid chromatograph 1 is comprised of a liquid chromatograph pump 2, a mobile phase reservoir 3, a sample injector 4, a separation column 5 and a tube 6. The mobile phase solvent in the mobile phase reservoir 3 is fed in its specified flow rate to the separation column 5 by the liquid chromatograph pump 2. Sample of mixture is fed into the mobile phase solvent by the sample injector 4 arranged between the liquid chromatograph pump 2 and the separation column 5. The sample of mixture reached to the separation column 5 is separated for every components under interaction with the packing material filled in the separation column 5. The separated sample substances are fed into an ion source 7 together with the mobile phase solvent.
Although there are several kinds of ion sources to be applicable, the case in which the ion source 7 operated under an electrospray will be described as one typical example of it. The sample component reached to the ion source 7 is fed into a metal tube 9 through a connector 8. As a high voltage of several kilo-volts is applied between the metal tube 9 and an electrode 10 arranged in opposition to the terminal end of the metal tube 9 by a high voltage power supply 11, electrospray may be produced from the terminal end of the metal tube 9 toward the opposing electrode 10. In this case, although a flow rate of solution capable of keeping a stable electrospray is several micro-litters per minute, a flow rate of solution fed from the liquid chromatograph 1 to the ion source 7 is normally 1 milli-litter per minute. In view of this fact, nebulization gas 13 supplied from a gas supplying tube 12 is flowed along the outer circumference of the metal tube 9 and then the aforesaid electrospray is assisted by the high speed gas flow. Since some ions related to the components in the sample are contained in liquid droplets generated by the electrospray, the liquid droplets are dried to enable ions of gaseous sample component to be attained.
The ions produced in this way are fed through an ion introduction aperture 14a arranged in the opposing electrode 10 into a differential pumping region 16 evacuated by a vacuum pumping system 15a, and further fed through an ion introduction aperture 14b into a vacuum region 17 evacuated by a vacuum pumping system 15b. Ions fed into the vacuum region 17 are focused by an ion focusing lens 18 composed of electrodes 18a, 18b and 18c, thereafter the ions are fed into the ion trap type mass analysis region 20. In addition, the gate electrode 21 is arranged in order to control injection of ions into the ion trap type mass analysis region 20.
The ion trap type mass analysis region 20 is comprised of endcap electrodes 19a, 19b and a ring electrode 19c. In FIG. 2 are indicated an amplitude of a high frequency voltage applied to the ring electrode 19c and a change in time of a control voltage applied to the gate electrode 21 only for a time in which a mass spectrum is taken once (a relation in variation of time in respect to a voltage applied to each of the electrodes shown in FIG. 2 is hereinafter called as a scan function).
As shown in FIG. 2, at first, a high frequency voltage is applied to the ring electrode 19c within an ion accumulating period 101 and then a potential for use in trapping the ions is formed within a space enclosed by the endcap electrodes 19a, 19b and the ring electrode 19c (hereinafter called as an ion trap space). In this case, when positive ions are analyzed, a control voltage applied to the gate electrode 21 is decreased (on) in order to enable the positive ions to pass through the gate electrode 21. Ions introduced into the vacuum region 17 are focused by the focusing lens 18, pass through the opening of the endcap electrode 19a and then incident into the aforesaid ion trap space.
Collision gas such as helium gas or the like is fed into the aforesaid ion trap space and it is kept at a pressure of about 1 mTorr or the like, so that ions injected into the aforesaid ion trap space strike against molecules of the aforesaid collision gas to lose their energy, wherein the ions are trapped into the ion trap space by the ion trap potential formed in the aforesaid ion trap space.
Then, an amplitude of the high frequency voltage applied to the ring electrode 19c in the scan period 102 is increased in sequence to perform a mass scanning. In this case, the control voltage applied to the gate electrode 21 is increased (off) to prevent the ions from passing through the gate electrode 21. As an amplitude of the high frequency voltage applied to the ring electrode 19c is gradually increased, an orbit of ions having a lower value (m/z) in which a mass (m) of ions is divided by an electrical charge (z) of ions becomes unstable and then the ions are discharged out of the mass analysis region 20 through the opening of the endcap electrode 19b. The discharged ions are detected by an ion sensor 22, its detected signal is sent to a data processing unit 24 through a signal line 23 and processed there.
Upon completion of the scan period 102, the high frequency voltage applied to the ring electrode 19c is turned off to diminish an ion trapping potential and further remove ions remained in the mass analysis region 20 (the residual ion eliminating period 103). Such a series of operations (ion accumulation 101, scanning 102 and residual ion eliminating 103) are performed in a repetitive manner, resulting in that the mass of samples fed in sequence from the liquid chromatograph 1 can be analyzed.
In addition, although not illustrated in FIG. 1, the liquid chromatograph 1, the ion source 7 and the ion trap mass analysis region 20 are controlled by a control system (including a power supply for controlling operation, a control circuit or a control software or the like /not shown).
The mass spectrometer having the aforesaid electrospray region and the ion trap type mass analysis region is already disclosed in Analytical Chemistry, 1991, Volume No. 63 and page 375, for example. In addition, a principle in operation of the ion trap type mass analysis region is already disclosed in the official gazette of U.S. Pat. No. 4,540,884 or the like.
In the case that a mass spectrum is attained under application of the mass spectrometer, a value m/z of the sample ion appeared on the spectrum (m is a molecular weight of ion and z is a charge number of ion) must be determined accurately. Due to this fact, the present mass spectrometer has a scale (an index) called as a mass marker and the value of m/z of ion is calculated by reading this mass marker. However, since this mass marker does not always display an accurate value, it must be confirmed sometimes whether or not the mass marker is correct. In the case that the mass marker is not correct, it is necessary to correct the mass marker (a so-called mass calibration).
The correction of the mass marker is carried out such that a peak position of ion intensity of substance (a standard sample) having a known value of m/z of ion to be generated is measured and its adjustment is performed so as to cause the ion to be detected at a position of a predetermined m/z value. As the standard sample, polyethylene glycol having a molecular structure of HO--(CH.sub.2 --CH.sub.2 --O).sub.n --H (n is an optional positive integer) is generally applied.
As polyethylene glycol is ionized under application of electrospray, peaks of protonated quasi-molecular ions are strongly observed. FIG. 3 shows a mass spectrum in the case that two kinds of commercial available polyethylene glycol having an average molecular weight of each of 200 and 600 are mixed to each other and applied as a standard sample, respectively. As apparent from FIG. 3, under application of polyethylene glycol causes a plurality of peak values to be observed at positions spaced apart by about 44 of molecular weights. Accordingly, under an application of the mixed polyethylene glycol as standard samples as described above, it is possible to correct the mass marker over a wide range.
In addition, in the official gazette of Japanese Patent Laid-Open No. Hei 3-116646 is disclosed a method for correcting the mass marker with water cluster ions being applied as a standard sample.
However, in accordance with the study performed by the present inventors and the like, it has become apparent that the following problems may occur if the aforesaid polyethylene glycol is used as the standard sample in the mass spectrometer having the ion trap type mass analysis region. That is, since the number of ions which can be accumulated at the ion trap type mass analysis region is less, accumulation of ions exceeding limit causes an electric field in the mass analysis region to be strained by a Coulomb repulsion force with a large amount of spatial electric charge generated under an increased density of accumulation of a large amount of ions, and a reduction in performance such as a reduction in a mass resolution or a shift of the value of m/z (a reduction in resolution or a mass shift) to be observed. Accordingly, if a correction of mass marker is carried out with polyethylene glycol of high concentration being applied as a standard sample, many kinds of quasi-molecular ions are generated at positions spaced apart by about 44 in molecular weights, resulting in that ions exceeding limit are accumulated within the mass analysis region and a reduction in performance as described above (reduction in resolution or mass shift) is generated.
Under a state in which the resolution is decreased or the mass shift is produced, it is apparent that the mass marker can be accurately corrected. In addition, when the mass marker is corrected under application of sample having a low concentration in order to weaken the aforesaid Coulomb repulsion force, it is possible to avoid the reduction in resolution or the mass shift, although an ion intensity is weak, resulting in that the number of count at the peak position is reduced and the peak position can not be determined accurately and the accurate mass calibration is difficult.
In addition, as the standard substances, if the value of m/z of the generated ions is known, any type of substances can be applied. However, in general, one type of substance mainly generates one type of ion only, so that in the case that the mass marker is corrected in a wide range of m/z value, many kinds of samples must be prepared and the operation becomes quite troublesome.